The present invention is directed to a new process for the production of pure urokinase.
Urokinase is a known enzyme which occurs in small amounts in the urine of mammals and accordingly also in human urine. Urokinase is an activator and serves to change plasminogen into plasmin. This enzyme in turn can dissolve fibrinous clots. Therefore urokinase preparations are valuable products in pharmaceutical medicine for the treatment, for example, of thromboembolisms.
There are known a number of processes for the isolation of urokinase from human urine and subsequent purification and concentration to a highly effective product. They consist of bringing the urine into contact with an adsorption agent and subsequently eluting the adsorbate. Then the crude urokinase is present in higher concentration in the extract. This extract in turn is worked up by means of various adsorption-elution and other separating processes so that finally there is obtained a highly concentrated and purified urokinase solution.
Known adsorption agents include for example calcium carbonate, barium sulfate, aluminum oxide, calcium phosphate, zinc hydroxide, activated carbon, hydrated aluminum silicates such as bentonite and kaolin, ion exchange silicates, molecular sieves as well as a number of other organic and inorganic materials.
In the meantime there have been proposed various affinity-chromatographic methods for the concentration and purification of urokinase. Most of these processes use for the affinitive adsorption solid matrices which e.g. contain the following compounds immobilized superficially: epsilon-aminocaproic acid and p-aminoenzamidine on Sepharose.RTM., antibody for urokinase on Sepharose, arginine on polyacrylamide resins, agmaline-epsiton-caproic acid on Sepharose.RTM., trypsin inhibitor on agarose, lysin or argenine on agcerose, trypsin inhibitor on Sepharose, urokinase inhibitor from human placenta on Sepharose and similar combinations.
On the other hand it is known to immobilize aprotinin on the surface of a solid matrix and to use the resin of this type which is prepared for the affinity-chromatographic separation and concentration of various compounds. However it is not known to use it with urokinase. For example in the Zeitschrift Biochimie 55, page 1323 (1973) there is described an affinity-chromatographic process for the concentration of trypsin on this kind of resin. A similar process is described in the same Zeitschrift in Volume 15, page 4 (1976). Furthermore there is described in the Journal of Physiological Chemistry 357, pages 1153 et seq. (1976) a process for the purification and characterization of human pancreaselastase. Finally note also the work in the Journal of Clinical Chemistry/Clinical Biochemistry, 15, pages 479 et seq. (1977) where there is described a process for the isolation of human kallikrein (callicrein).